Introduction
Biofertilizers such as Trichoderma viride have emerged as a sustainable alternative to improve plant nutrition, increase tolerance to stress factors,and reduce reliance on syn-thetic fertilizers.Within this group,species of the genus Trichoderma,particularly Trichoderma viride,stand out for their ability to promote plant growth,enhance soil fertility,and biologically control soil pathogens,making them key tools for developing more resilient and sustainable agricultural systems. In addition, other plant growth-promoting rhizobacteria(PGPR) and arbuscular mycorrhizal fungi(AMF) have shown high potential for improving crop growth and yield, including in wheat. PGPR, such as those from the genus Azospirillum, stimulate root and shoot development through the production of phytohormones like indole-3-acetic acid and gibberellins, while AMF, primarily from the genus Glomus, enhance water and phosphorus uptake and increase tolerance to abiotic stress. Recent studies have reported that the coinoculation of PGPR and AMF significantly increases root length, surface area, and dry biomass.
Empirical evidence supports the potential of these biofertilizers in wheat and other grasses, showing yield and grain quality improvements. Grageda-Cabrera et al. demon-strated that AMF inoculation in wheat increased yields by up to 1.2 t ha-1 and improved nitrogen use efficiency by 11%. Similarly, Cisse et al. found that combining biofertilizers with manure reduced the need for chemical fertilizers by up to 50% without compromising yields and, in some seasons, even increased yields by 6.8%to 12.4%. Likewise, Bhawana et al., working with pearl millet(Pennisetum glaucum) in a pearl millet–wheat cropping system, reported significant improvements in yield and morphophysiological traits with the combined application of liquid biofertilizers and standard fertilization— results that can be extrapolated to wheat systems Similarly, Zaheer et al. demonstrated that the cytokinin-producing strain A.brasilense RA-17 enhanced the net assimilation rate,leaf area,and grain yield of wheat, evidencing the positive physiological influence of biofertilizers on C3 crops.
In parallel, fish-derived liquid inputs (hydrolysates/ferments) provide readily avail-able amino acids, peptides, micronutrients, and bioactive compounds that can stimulate early seedling vigor, microbial activity, and nutrient cycling. Furthermore, research on wheat crop has confirmed significant gains in growth and yield through the application of liquid biofertilizers containing Azospirillum and Azotobacter. In Egypt, El-Sorady et al. reported a 14% increase in grain yield with Azotobacter inocu-lation, while Kaur et al. observed the maximum yield with a combined inoculation with Streptomyces sp.
Materials and Methods
Study Site
The study was conducted during the 2023–2024 growing season at the Sulluscocha Experimental Annex of the Baños del Inca Agricultural Experimental Station(INIA),located in the district of Llacanora,Cajamarca Province,Peru(7◦12′8.06′′S,78◦22′18.10′′W;2984 m a.s.l.)(Figure1).
Meteorological variables were obtained from SENAMHI.During the experimental pe-riod,recorded temperatures ranged from 7.9℃ to 22.5℃,with mean daily values between 14.3℃ and 18.6℃.The highest monthly precipitation was observed in February,reaching 120.3 mm. During the 2023–2024 agricultural campaign,rainfall in the experimental area remained within the typical range for the region,with no extreme events affecting crop development. Relative humidity was higher in the rainy months, reaching a maximum of 82% in January, and progressively decreasing to a minimum of 66% in August and September. The relative humidity exhibited higher values during the rainy season, with a maximum of 82% observed in January, and subsequently declined to a minimum of 66% during August and September. On average, the relative humidity throughout the growing season was approximately 74.3%(Figure2).
Soil Physicochemical Properties
Before establishing the experiment,composite soil samples were collected from a depth of 0–40 cm and analyzed at the Soil Laboratory of the Baños del Inca Agricultural Experimental Station(INIA). The evaluated soil parameters included soil texture, pH, electrical conductivity, organic matter content, total nitrogen(N), available phosphorus(P), available potassium(K) and the concentration of exchangeable cations(Table1).
| Parameter | Result |
| pH | 5.3 |
| Electrical conductivity (S m−1) | 11.5 |
| Organic matter (%) | 3.8 |
| Total nitrogen (%) | 0.19 |
| Available phosphorus (mg kg−1) | 15.2 |
| Available potassium (mg kg−1) | 233.6 |
| Texture | Clay loam |
Experimental Design and Agronomic Management
The field trial followed a 5×5 Latin square design,totaling 25 experimental units arranged in five rows and five columns to control orthogonal gradients of soil fertility and microclimate.This design was employed to minimize experimental error associated with field heterogeneity in two directions, ensuring that each treatment was evaluated once per row and once per column, thus statistically controlling for positional effects.
Treatments were randomly assigned within each row and column following a complete randomization scheme to avoid positional bias.Each experimental unit consisted of a 4.8 m2 plot composed of four rows, each 4 m in length and spaced 0.30 m apart; plots were separated by 1 m alleys to facilitate access and management. The net experimental area was 120 m2, and the gross area, including alleys,was 156 m2.Sowing was conducted on 20 February 2024, within the recommended planting window for the site, under rainfed conditions. In each plot, four rows were established by continuous seeding along the furrows using 70 g of seed per plot.
Inoculation treatments were applied once at sowing,and each inoculant was handled independently to avoid cross-contamination. No mixed inoculations were performed; each treatment corresponded to a single inoculation applied separately at the time of seeding,using sterile equipment and separate containers for each microbial material to ensure experimental integrity.
Five treatments were evaluated. TR1 was an absolute control without mineral fertilization or bioinoculants. TR2 received 100%mineral fertilization according to soil-test recommendations; nitrogen was split-applied, with 50% at sowing and 50%at tillering. The total fertilizer inputs per plot were 210 g of diammonium phosphate(DAP), 120 g of potassium chloride(KCl), and 370 g of urea, with no inoculation applied. TR3–TR5 involved pre-sowing seed inoculation by direct impregnation with specific biofertilizers: Seeds in TR1 (absolute control) and TR2 (full mineral fertilization) did not receive any form of liquid treatment or simulated inoculation; they were sown completely dry. TR3 used A.brasilense, TR4 used Trichomax (Trichoderma viride) ,and TR5 used an anchovy (Engraulis ringens)-based liquid biofertilizer produced via anaerobic biofermentation. After inoculation(TR3–TR5), seeds were shade-air-dried to ensure uniform coating and inoculant viability prior to sowing.
Results
Correlation Analysis
The correlation matrix revealed coherent associations among agronomic and physio-logical traits(Figure3). Plant height was positively correlated with root dry mass(r=0.63), thousand-grain weight(r=0.58), and spike length(r=0.59), and root dry mass showed a positive association with grain yield(r=0.43). Thousand-grain weight displayed moderate to strong correlations with grains per spike(r=0.69) and spike length(r=0.54), underscoring its relevance to yield potential. The strongest relationship was between grains per spike and spike length(r=0.88), reinforcing the structural linkage between spike morphology and grain number. By contrast,grain yield exhibited generally weak correlations with most traits aside from root mass, and test weight showed a slight negative association with grain yield (r=-0.24).
Agronomic and Physiological Traits
Plant Height(cm)
Plant height was lowest in the unfertilized, uninoculated control (TR1:53.05±3.99 cm), while the highest means were observed in the anchovy-based biofertilizer(TR5:57.77±4.07 cm) and mineral fertilization (TR2:57.06±4.06 cm) treatments. Intermediate values occurred with seed inoculation by A.brasilense (TR3:55.74±4.04 cm) and Trichoderma (TR4:54.27±4.01 cm). Compared to the control, plant height increased by 7.6%under mineral fertilization, 8.9%with the anchovy-based biofertilizer, 5.1%with A.brasilense, and 2.3%with Trichoderma.Collectively, these results indicate modest but consistent improvements in plant height with both chemical fertilization and biofertilizer application relative to the untreated control (Figure4A,Table2).
| Treatment | Spike Length (cm) | Number of Grains per Spike | Thousand Grain Weight (g) | Root Dry Mass (g) | Plant Height (cm) | Test Weight (kg hL−1) | Grain Yield (t ha−1) |
| Mean ± E.E | Mean ± E.E | Mean ± E.E | Mean ± E.E | Mean ± E.E | Mean ± E.E | Mean ± E.E | |
| TR1 | 5.9 b ± 0.58 | 24 a ± 3.22 | 42.24 a ± 1.83 | 2.34 c ± 1.21 | 53.05 a ± 3.99 | 75.28 a ± 4.33 | 0.6 c ± 0.47 |
| TR2 | 6.9 a ± 0.56 | 30 a ± 3.43 | 42.66 a ± 2.30 | 4.90 a ± 1.77 | 57.06 a ± 4.06 | 68.10 a ± 4.24 | 1.2 a ± 0.79 |
| TR3 | 6.5 ab ± 0.88 | 29 a ± 3.40 | 42.54 a ± 2.83 | 4.00 ab ± 1.61 | 55.74 a ± 4.04 | 70.58 a ± 4.27 | 0.9 ab ± 0.64 |
| TR4 | 6.1 ab ± 0.87 | 23 a ± 3.18 | 41.90 a ± 2.52 | 2.96 bc ± 1.38 | 54.27 a ± 4.01 | 73.88 a ± 4.32 | 0.8 bc ± 0.59 |
| TR5 | 6.9 a ± 0.40 | 28 a ± 3.37 | 42.00 a ± 3.25 | 3.20 bc ± 1.44 | 57.77 a ± 4.07 | 72.78 a ± 4.30 | 0.8 bc ± 0.59 |
Spike Lenght(cm)
Spike length was greatest under mineral fertilization(TR2:6.9±0.56 cm)and the anchovy-based liquid biofertilizer (TR5:6.9±0.40 cm),followed by A brasilense(TR3: 6.5±0.88 cm)and Trichoderma(TR4:6.1±0.87 cm),with the shortest spikes in the un-treated control(TR1:5.9±0.58 cm).Relative to the control,spike length increased by 16.9%under mineral fertilization,16.9%with the anchovy-based biofertilizer,10.2%with A.brasilense,and 3.4%with Trichoderma.These patterns indicate that both chemical fertil-ization and the fish-derived biofertilizer were associated with longer spikes relative to the control.(Figure4B,Table2).
Root Dry Mass(g)
Root dry mass differed significantly among treatments(p<0.05).Mineral fertiliza-tion achieved the highest mean(TR2:4.90±1.77 g),followed by seed inoculation with A.brasilense(TR3:4.00±1.61 g).Intermediate values were observed for Trichoderma(TR4:2.96±1.38 g)and the anchovy-based liquid biofertilizer(TR5:3.20±1.44 g),whereas the untreated control showed the lowest root mass(TR1:2.34±1.21 g).Relative to the control,root biomass increased by 109.4%under mineral fertilization,70.9%with A.brasilense,36.8%with the anchovy-based biofertilizer,and 26.5%with Trichoderma.Overall,both min-eral fertilization and A.brasilense inoculation markedly enhanced belowground biomass compared to the untreated control(Figure4C,Table2).
Number of Grains per Spike
The highest number of grains per spike was observed under mineral fertilization(TR2:30±3.43) and A brasilense inoculation(TR3:29±3.40). Intermediate means occurred with the anchovy-based biofertilizer(TR5:28±3.37) and the untreated control(TR1:24±3.22), while the lowest value was recorded with Trichoderma(TR4:23±3.18).Relative to the control,the number of grains per spike increased by 25.0%under min-eral fertilization,20.8%with A.brasilense,and 16.7%with the anchovy-based biofertilizer, whereas Trichoderma showed a slight reduction of 4.2%. Although some treatments showed slight numerical increases in grain number per spike,these differences were not statistically significant (Figure5A,Table2).
Test Weight(kg hL-1)
Test weight was highest in the untreated control(TR1:75.28±4.33 kg hL-1), followed by Trichoderma(TR4:73.88±4.32 kg hL-1) and the anchovy-based biofer-tilizer(TR5:72.78±4.30 kg hL-1), with slightly lower means for A.brasilense(TR3:70.58±4.27 kg hL-1) and mineral fertilization(TR2:68.10±4.24 kg hL-1). Overall,grain physical quality appeared uniform across treatments,as all means fell within the same statistical group(Figure5B,Table2).
Thousand Grain Weight(g)
Thousand-grain weight showed minimal variation among treatments.The highest mean was recorded under mineral fertilization(TR2:42.66±2.30 g), closely followed by A.brasilense(TR3:42.54±2.83 g), the anchovy-based biofertilizer(TR5:42.00±3.25 g), and the untreated control(TR1:42.24±1.83 g), with the lowest value under Tricho-derma(TR4:41.90±2.52 g). Overall, thousand-grain weight remained stable across treatments, indicating no appreciable effect of fertilization or inoculation on this grain physical attribute(Figure5C,Table2).
Grain Yield(t ha-1)
Grain yield was highest under mineral fertilization(TR2:1.20±0.79 t ha-1) representing a 100%increase compared with the untreated control(TR1:0.60±0.47 t ha-1), which had the lowest mean.Intermediate yields were observed with A.brasilense(TR3:0.90±0.64 t ha-1), Trichoderma(TR4:0.85±0.59 t ha-1), and the anchovy-based biofertiizer (TR5:0.81±0.59 t ha-1), corresponding to yield improvements of 50.0%, 41.7%, and 35.0%, respectively, relative to the control(Figure5D,Table2).
Discussions
Our field results demonstrate that biofertilizer inoculation can significantly enhance wheat performance in high-altitude rainfed systems.The application of microbial and or-ganic biofertilizers—A brasilense, Trichoderma viride, and an anchovy-based liquid fertilizer—improved both physiological traits and yield outcomes of wheat(cv.INIA 405) under realistic farming conditions. In particular, A.brasilense seed inoculation stimulated vigorous belowground growth, evidenced by a marked increase in root biomass(relative to the control). This is consistent with the well-documented root-promoting activity of Azospirillum, which produces phytohormones (e.g.,auxins,gibberellins) that spur root development and thereby expand the plant’s capacity for water and nutrient uptake. A more developed root system in the inoculated plants likely underpinned their improved shoot growth and yield. Indeed, A.brasilense treatment in our trial raised grain yield to~0.90 t ha-1, about a 50% increase over the unfertilized control(0.60 t ha-1) and approaching the yield obtained with full NPK fertilization(~1.2 t ha-1). Because inoculated treatments received no mineral fertilizers yet achieved~70–75%of the yield of NPK fertilization, our results support biofertilizers as complementary inputs that can reduce mineral fertilizer requirements under low-input conditions, not as full substitutes for mineral nutrient supply.
The anchovy-derived biofertilizer also showed notable aboveground benefits:it significantly increased spike length and grain number per spike compared to the control,indicating an improvement in spike architecture. This finding aligns with reports that fish protein hydrolysate biostimulants can promote the development of larger wheat ears with more grains. Trichoderma viride inoculation yielded a more moderate improvement in grain yield and did not markedly change root or spike metrics in our study;however,the positive yield response suggests that Trichoderma’s benefits may manifest through subtler mechanisms such as enhanced nutrient mobilization or stress alleviation(even without dramatic changes in morphology). In sum,all three biofertilizers boosted wheat growth and productivity relative to unfertilized plants,with A.brasilense showing the strongest effect on root biomass and the fish-based fertilizer most enhancing spike traits.
Although the present experiment focused primarily on plant responses,several well-documented soil and rhizosphere mechanisms help explain the observed yield and biomass improvements under Azospirillum, Trichoderma, and anchovy-based biofertilizer treatments.For Trichoderma, multiple studies demonstrate that this fungus enhances root development,nutrient mobilization,and physiological resilience through mechanisms such as improved rhizospheric enzyme activities,enhanced nutrient solubilization, and modulation of plant stress responses.For example, Trichoderma viride has been shown to significantly increase plant height,root length,and spike length in wheat, especially when combined with organic substrates such as humic acids. Other studies report increased yield components(up to+36.5%grain yield) associated with enhanced nutrient acquisition and improved root physiology.In addition, Trichoderma viride can activate host antioxidant enzymes such as catalase, peroxidases, and superoxide dismutase, thereby reducing oxidative stress and improving plant health under biotic challenges. These rhizosphere and physiological effects are consistent with the moderate but positive yield responses we observed for Trichoderma viride inoculation.
The comparatively weaker performance of Trichoderma viride in our field trial may reflect the interaction between its physiological mechanisms and the edaphic constraints of high-altitude soils. Although Trichoderma is well known to promote plant growth through auxin and gibberellin production,enhancement of nutrient utilization efficiency, and stimulation of root-associated enzymatic activities, the expression of these functions is strongly conditioned by environmental factors. According to recent comprehensive syntheses, Trichoderma relies on multiple modes of action such as competition, antibiosis, mycoparasitism, secretion of cell-wall-degrading enzymes, volatile secondary metabolites,and induction of systemic resistance to support plant performance. However,the activity of these mechanisms decreases under suboptimal pH, low temperatures, or nutrient-limited soils,which can reduce conidial germination,metabolic activity,and the production of key hy-drolytic enzymes.Such environmental sensitivity may have constrained the ability of Trichoderma viride to colonize roots effectively or to express its full biostimulatory potential under our high-Andean conditions,thereby explaining its more moderate yield response compared with A.brasilense and the anchovy-based biofertilizer.
A.brasilense also exerts well-characterized mechanistic effects on wheat that align with our observations. Experimental evidence shows that A.brasilense enhances nitrogen uptake and accumulation, increases grain protein concentration, and stimulates root proliferation through hormonal pathways such as auxin production. Field studies further demonstrate that inoculation improves nutrient uptake efficiency across macro-and micronutrients and can increase wheat yields by 13–31%depending on the strain and environmental conditions. More recent trials confirm that A.brasilense enhances agronomic N-use efficiency, increases N recovery,and raises yield by>10%even under varying N fertilization regimes.
Beyond nutrient uptake, the hormonal activity of A brasilense provides an additional mechanistic explanation for the strong root proliferation observed in our trial. A hallmark trait of Azospirillum is its ability to synthesize indole-3-acetic acid(IAA), the primary auxin regulating cell division, elongation, and root system architecture. Recent evidence demonstrates that IAA biosynthesis in A.brasilense is highly plastic and responsive to environmental cues: daylight, osmotic stress(PEG), abscisic acid, salicylic acid, chitosan, and fungal elicitors(e.g.,Fusarium oxysporum) all significantly increase IAA production and ipdC gene expression. This suggests that under stressful highland rainfed conditions,
Crucially,the yield improvements from biofertilization did not come at the cost of grain quality in our experiment. Grain physical parameters,including thousand-grain weight and test weight,remained statistically similar across all treatments.The 1000-grain weight hovered around~42 g in both biofertilized and control plots,and test weight was around 68.10–75.28 kg hL-1 for all,indicating that grains from biofertilizer-treated plants were as well-filled and dense as those from untreated or fully fertilized plants.This stability in grain size and density suggests that the additional grains produced under biofertilizer treatments were not smaller or shriveled—an encouraging outcome implying no trade-off between yield quantity and quality.In some cases,biofertilizers may even enhance certain quality aspects:for instance,the fish protein hydrolysate used by Mironenko et al. led to slight increases in wheat grain protein(about+2%protein content and+5%gluten content) alongside a~5%yield boost. We did not measure protein content in our study,but maintaining high test weight and kernel weight is itself a positive sign of grain quality. Overall,our findings concur with the notion that biofertilizers primarily increase yield by increasing the number of fertile spikes or grains(via improved growth and nutrient uptake), rather than by markedly altering individual grain mass. Even where modest increases in grain weight have been observed with PGPR or fungal inoculants,the dominant effect remains an increase in grain number per area. The use of these biofertilizers improved yield components(spike size,grain number) without detrimental effects on grain filling.
From an agronomic and ecological perspective,these results are highly promising. Biofertilizers are not a complete replacement for mineral fertilizers, but our study illustrates that they can reduce the requirement for synthetic inputs while improving crop performance—a win-win for sustainability. Farmers in highland areas could utilize products like Azospirillum and Trichoderma inoculants or fish-based fertilizers to
boost their wheat yields and stability,cutting down on costly chemical fertilizer usage.This not only has economic benefits(lower input costs, potentially higher net profits)but also environmental ones.Reduced application of chemical N/P fertilizers means lower risk of nitrogen leaching and runoff,and a smaller carbon footprint associated with fertilizer production and use.By leveraging naturally occurring soil microbes and organic nutrient sources,this approach aligns with the principles of agroecological intensification—enhancing soil health and fertility through biological means rather than solely through industrial inputs. Notably,A.brasilense and Trichoderma viride are both benign organisms that pose minimal risk to the environment,making them safe and sustainable tools for crop management.Their use can also contribute to long-term soil quality(e.g.,through better root growth and organic matter inputs from increased residue),thereby fostering a more resilient agroecosystem in the face of climatic stresses.
Conclusions
This study provides the first field-based evidence from the Peruvian Andes that biofertilizers can substantially enhance wheat performance under rainfed,high-altitude conditions.Unlike most previous trials conducted in irrigated or temperate regions,these results were obtained under the challenging agroecological context of the Andes,where soils are marginal and synthetic inputs are limited.The demonstrated improvements in root biomass,spike architecture,and yield highlight the potential of A.brasilense,Trichoderma viride,and fish-based organic fertilizers to strengthen crop productivity within these resource-constrained environments.Importantly,these biofertilizers should be viewed as complementary components of integrated nutrient management rather than complete substitutes for mineral fertilizers.Their application can improve nutrient use efficiency and soil biological activity,allowing partial reductions in synthetic fertilizer use without compromising grain quality.This integrative approach offers a realistic pathway toward more sustainable,low-input wheat production systems across the Andean highlands,combining traditional practices with modern microbial and organic technologies to enhance both yield and environmental resilience.
To support broader adoption of biofertilizers in Andean agriculture,targeted pro-motion strategies are essential. To ensure consistent field performance, we recommend that extension programs provide clear protocols for seed inoculation(e.g.,dosage,dilution,drying time,and storage conditions)and promote the co-application of biofertilizers with minimal starter mineral fertilization under highly depleted soils.
If you are interested in bio-fertilizers or fish fertilizers, you are welcome to contact the Dora team.
